1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is the hormonal metabolite of vitamin D3 which is postulated to influence specific biochemical events through receptor-mediated actions on the cellular genome. We propose to utilize specific immunobgic probes (monoclonal antibodies) against the 1,25 (OH)2O3 receptor to elucidate this protein's biologic and molecular function in both chick and cultured cell models. The nuclear translocation hypothesis will be tested cytologically through immunohistochemical techniques designed to ascertain the protein's subcellular distribution in chick intestine. The cellular distribution of receptor will also be determined, enabling subsequent correlations to be made with existing autoradiographic (1,25(OH)2D3) and histochemical (D-dependent calcium-binding protein) data. Several aspects of receptor biology will be explored through application of a receptor radioimmunoassay: 1) effect of 1,25(OH)2D3 levels on nuclear localization of receptors; 2) the nature of nuclear-receptor interaction; 3) the biochemical fate of receptors following binding to genomic constituents; and 4) regulation of cellular receptor concentration. Determination of the receptor's biochemical function will be approached through molecular characterization. The receptor will be purified by immunoaffinity chromatography, and then chemically characterized by proteolytic digestion experiments and amino acid analysis. Bio-molecular properties of the intact molecule will also be evaluated through immunologic examination of 1) ligand-binding domains (1,25(OH)2D# and DNA); and 2) the molecule's potential for enzymatic activity (phosphorylation and acetylation). These techniques will then be adapted to similar evaluations of 1,25(OH)2D3 receptors from cultured-cell models (mouse fibroblasts (3T6) and human intestinal cells (IN-407)). Most importantly, these procedures will also be used to evaluate receptor defects in human fibroblasts derived from patients lacking vitamin D responsiveness. The present application of immunologic techniques represents a new approach towards delineating the molecular action of vitamin D3, an action which relates not only tothe regulation of mineral metabolism, but to a host of other newly identified biologic responses.